The root of Polygala tenuifolia WILLD (PT) (Japanese name: Onji) is a well-known traditional Chinese medicine used as an expectorant, a tonic, a tranquillizer and an anti-dementia drug. However, basic researches of the anti-dementia effects are very few in spite of PT being generally recognized as a good herbal drug for amnesia. It was reported that 80% ethanol extract of PT and the methanol extract of PT reverse scopolamine-induced cognitive impairments in rats. 1,2) Acetylcholinestrase (AChE) activity is inhibited in vitro by 80% ethanol extract of PT.1,3) Onjisaponin F, a constituent in PT, increases cholineacetyltransferase (ChAT) mRNA level in rat basal forebrain cell, and. Taken together, transient enhancement of cholinergic systems by PT may be involved in the memory improvement seen in like the scopolamine model. However, practical dementia is caused by irreversible neuronal damage involving neuronal death, neuritic atrophy and synaptic loss. Especially in case of Alzheimer's disease, neuritic atrophy and synapse loss are earlier events than neuronal death are, and are critical for memory disorder. [4][5][6][7][8] Previous our studies suggested that compounds which showed synaptic regeneration activity in cultured neurons were also active for memory impairment in vivo using Alzheimer's disease model mice.9-11) Although protective effects of PT on neuronal death were shown in amyloid b (Ab), glutamate or NMDA-induced neuronal damage, effects of PT on atrophy of neuritis and synaptic loss never have been investigated yet.1,12) Therefore, we aimed in this study to characterize activities of PT under Ab-induced neuritic damage.
MATERIALS AND METHODS
MaterialsThirty gram of roots of Polygala tenuifolia WILLD (PT) was extracted with 600 ml of water at 100°C for 40 min. This crude drug was purchased from Tochimoto Tenkaido (Osaka, Japan). The decoction was evaporated under reduced pressure and freeze-dried of extract powder (yield: 8.02 g). The extract was then dissolved in sterilized distilled water at 1000 times concentrations of final concentrations. Ab (25-35) (Sigma, Saint Louis, MO, U.S.A.) was dissolved in sterile distilled water at a concentration of 5 mM, and was incubated at 37°C for 3 d to allow fibril formation. [Gly 14 ]-Humanin was purchased from Phoenix Pharmaceuticals (Belmont, CA, U.S.A.) as a positive control for neuronal protection. A monoclonal antibody to phosphorylated neurofilament-H (NF-H) was purchased from Sternberger Monoclonals (Lutherville, MD, U.S.A.). A monoclonal antibody to microtubule-associated protein 2 (MAP2), an antiserum to MAP2, polyclonal antibody to MAP2 and a monoclonal antibody to synaptophysin were purchased from Chemicon (Temecula, CA, U.S.A.). Alexa Fluor 488-conjugated goat anti-mouse IgG and Alexa Fluor 546-conjugated goat antirabbit IgG were purchased from Molecular Probes (Eugene, OR, U.S.A.). Aqua Poly Mont was purchased from Polyscience (Warrington, PA, U.S.A.).Primary Culture Embryos were removed from pregnant Sprague Dawley rats (Japan SLC, Shizuoka, J...